Mouse erythroid cells express multiple putative RNA helicase genes exhibiting high sequence conservation from yeast to mammals

RNA secondary structure is a critical determinant of RNA function in ribosome assembly, pre-mRNA splicing, mRNA translation and RNA stability. The ‘DEAD/H’ family of putative RNA helicases may help regulate these processes by utilizing intrinsic RNA-dependent ATPase activity to catalyze conformational changes in RNA secondary structure. To investigate the repertoire of DEAD/H box proteins expressed in mammals, we used PCR techniques to clone from mouse erythroleukemia (MEL) cells three new DEAD box cDNAs with high similarity to known yeast (Saccharomyces cerevisiae) genes. mDEAD2 and mDEAD3 (mouse DEAD box proteins) are >95% identical to mouse PL10 but exhibit differential tissue-specific expression patterns; mDEAD2 and mDEAD3 are also approx. 70% identical (at the aa level) to yeast DED1 and DBP1 proteins. Members of this DEAD box subclass contain C-terminal domains with high content of Arg, Ser, Gly and Phe, reminiscent of the RS domain in several Drosophila and mammalian splicing factors. mDEAD5 belongs to a second class related to translation initiation factors from yeast (TIF1/TIF2) and mammals (eIF-4A); this class contains a novel conserved peptide motif not found in other DEAD box proteins. Northern blotting shows that mDEAD5 is differentially expressed in testis vs. somatic tissues. Thus, mouse erythroid cells produce two highly conserved families of putative RNA helicases likely to play important roles in RNA metabolism and gene expression.     

Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses

A number of RNA-containing viruses such as hepatitis C (HCV) and poliovirus (PV) that infect human beings and cause serious diseases use a common mechanism for synthesis of viral proteins, termed internal ribosome entry site (IRES)-mediated translation. This mode of translation initiation involves entry of 40S ribosome internally to the 5' untranslated region (UTR) of viral RNA. Cap-dependent translation of cellular mRNAs, on the other hand, requires recognition of mRNA 5' cap by the translation machinery. In this review, we discuss two inhibitors that specifically inhibit viral IRES-mediated translation without interfering with cellular cap-dependent translation. We present evidence, which suggest that one of these inhibitors, a small RNA (called IRNA) originally isolated from the yeast Saccharomyces cerevisiae, inhibits viral IRES-mediated translation by sequestering both noncanonical transacting factors and canonical initiation factors required for IRES-mediated translation. The other inhibitor, a small peptide from the lupus autoantigen La (called LAP), appears to block binding of cellular transacting factors to viral IRES elements. These results suggest that it might be possible to target viral IRES-mediated translation for future development of therapeutic agents effective against a number of RNA viruses including HCV that exclusively use cap-independent translation for synthesis of viral proteins.     

Functional roles of DExD/H-box RNA helicases in Pre-mRNA splicing

Splicing of precursor mRNA takes place via two consecutive steps of transesterification catalyzed by a large ribonucleoprotein complex called the spliceosome. The spliceosome is assembled through ordered binding to the pre-mRNA of five small nuclear RNAs and numerous protein factors, and is disassembled after completion of the reaction to recycle all components. Throughout the splicing cycle, the spliceosome changes its structure, rearranging RNA-RNA, RNA-protein and protein-protein interactions, for positioning and repositioning of splice sites. DExD/H-box RNA helicases play important roles in mediating structural changes of the spliceosome by unwinding of RNA duplexes or disrupting RNA-protein interactions. DExD/H-box proteins are also implicated in the fidelity control of the splicing process at various steps. This review summarizes the functional roles of DExD/H-box proteins in pre-mRNA splicing according to studies conducted mostly in yeast and will discuss the concept of the complicated splicing reaction based on recent findings.     

An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast

The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.     

Functions of DEAD-box proteins in bacteria: Current knowledge and pending questions

DEAD-box proteins are RNA-dependent ATPases that are widespread in all three kingdoms of life. They are thought to rearrange the structures of RNA or ribonucleoprotein complexes but their exact mechanism of action is rarely known. Whereas in yeast most DEAD-box proteins are essential, no example of an essential bacterial DEAD-box protein has been reported so far; at most, their absence results in cold-sensitive growth. Moreover, whereas yeast DEAD-box proteins are implicated in virtually all reactions involving RNA, in E. coli (the bacterium where DEAD-box proteins have been mostly studied) their role is limited to ribosome biogenesis, mRNA degradation, and possibly translation initiation. Plausible reasons for these differences are discussed here.     

An intricate RNA structure with two tRNA-derived motifs directs complex formation between yeast aspartyl-tRNA synthetase and its mRNA

Accurate translation of genetic information necessitates the tuned expression of a large group of genes. Amongst them, controlled expression of the enzymes catalyzing the aminoacylation of tRNAs, the aminoacyl-tRNA synthetases, is essential to insure translational fidelity. In the yeast Saccharomyces cerevisiae, expression of aspartyl-tRNA synthetase (AspRS) is regulated in a process necessitating recognition of the 5' extremity of AspRS messenger RNA (mRNA(AspRS)) by its translation product and adaptation to the cellular tRNA(Asp) concentration. Here, we have established the folding of the approximately 300 nucleotides long 5' end of mRNA(AspRS) and identified the structural signals involved in the regulation process. We show that the regulatory region in mRNA(AspRS) folds in two independent and symmetrically structured domains spaced by two single-stranded connectors. Domain I displays a tRNA(Asp) anticodon-like stem-loop structure with mimics of the aspartate identity determinants, that is restricted in domain II to a short double-stranded helix. The overall mRNA structure, based on enzymatic and chemical probing, supports a three-dimensional model where each monomer of yeast AspRS binds one individual domain and recognizes the mRNA structure as it recognizes its cognate tRNA(Asp). Sequence comparison of yeast genomes shows that the features within the mRNA recognized by AspRS are conserved in different Saccharomyces species. In the recognition process, the N-terminal extension of each AspRS subunit plays a crucial role in anchoring the tRNA-like motifs of the mRNA on the synthetase.     

Human RNA 5'-kinase (hClp1) can function as a tRNA splicing enzyme in vivo

Yeast and human Clp1 proteins are homologous components of the mRNA 3′-cleavage-polyadenylation machinery. Recent studies highlighting an association of human Clp1 (hClp1) with tRNA splicing endonuclease and an intrinsic RNA-specific 5′-OH polynucleotide kinase activity of hClp1 have prompted speculation that Clp1 might play a catalytic role in tRNA splicing in animal cells. Here, we show that expression of hClp1 in budding yeast can complement conditional and lethal mutations in the essential 5′-OH RNA kinase module of yeast or plant tRNA ligases. The tRNA splicing activity of hClp1 in yeast is abolished by mutations in the kinase active site. In contrast, overexpression of yeast Clp1 (yClp1) cannot rescue kinase-defective tRNA ligase mutants, and, unlike hClp1, the purified recombinant yClp1 protein has no detectable RNA kinase activity in vitro. Mutations of the yClp1 ATP-binding site do not affect yeast viability. These findings, and the fact that hClp1 cannot complement growth of a yeast clp1Δ strain, indicate that yeast and human Clp1 proteins are not functional orthologs, despite their structural similarity. Although hClp1 can perform the 5′-end-healing step of a yeast-type tRNA splicing pathway in vivo, it is uncertain whether its kinase activity is necessary for tRNA splicing in human cells, given that other mammalian counterparts of yeast-type tRNA repair enzymes are nonessential in vivo.     

The structural basis of Edc3- and Scd6-mediated activation of the Dcp1:Dcp2 mRNA decapping complex

The Dcp1:Dcp2 decapping complex catalyses the removal of the mRNA 5' cap structure. Activator proteins, including Edc3 (enhancer of decapping 3), modulate its activity. Here, we solved the structure of the yeast Edc3 LSm domain in complex with a short helical leucine-rich motif (HLM) from Dcp2. The motif interacts with the monomeric Edc3 LSm domain in an unprecedented manner and recognizes a noncanonical binding surface. Based on the structure, we identified additional HLMs in the disordered C-terminal extension of Dcp2 that can interact with Edc3. Moreover, the LSm domain of the Edc3-related protein Scd6 competes with Edc3 for the interaction with these HLMs. We show that both Edc3 and Scd6 stimulate decapping in vitro, presumably by preventing the Dcp1:Dcp2 complex from adopting an inactive conformation. In addition, we show that the C-terminal HLMs in Dcp2 are necessary for the localization of the Dcp1:Dcp2 decapping complex to P-bodies in vivo. Unexpectedly, in contrast to yeast, in metazoans the HLM is found in Dcp1, suggesting that details underlying the regulation of mRNA decapping changed throughout evolution.     

Divergent RNA binding specificity of yeast Puf2p

PUF proteins bind mRNAs and regulate their translation, stability, and localization. Each PUF protein binds a selective group of mRNAs, enabling their coordinate control. We focus here on the specificity of Puf2p and Puf1p of Saccharomyces cerevisiae, which copurify with overlapping groups of mRNAs. We applied an RNA-adapted version of the DRIM algorithm to identify putative binding sequences for both proteins. We first identified a novel motif in the 3' UTRs of mRNAs previously shown to associate with Puf2p. This motif consisted of two UAAU tetranucleotides separated by a 3-nt linker sequence, which we refer to as the dual UAAU motif. The dual UAAU motif was necessary for binding to Puf2p, as judged by gel shift, yeast three-hybrid, and coimmunoprecipitation from yeast lysates. The UAAU tetranucleotides are required for optimal binding, while the identity and length of the linker sequences are less critical. Puf1p also binds the dual UAAU sequence, consistent with the prior observation that it associates with similar populations of mRNAs. In contrast, three other canonical yeast PUF proteins fail to bind the Puf2p recognition site. The dual UAAU motif is distinct from previously known PUF protein binding sites, which invariably possess a UGU trinucleotide. This study expands the repertoire of cis elements bound by PUF proteins and suggests new modes by which PUF proteins recognize their mRNA targets.     

mRNAs encoding polarity and exocytosis factors are cotransported with the cortical endoplasmic reticulum to the incipient bud in Saccharomyces cerevisiae

Polarized growth in the budding yeast Saccharomyces cerevisiae depends upon the asymmetric localization and enrichment of polarity and secretion factors at the membrane prior to budding. We examined how these factors (i.e., Cdc42, Sec4, and Sro7) reach the bud site and found that their respective mRNAs localize to the tip of the incipient bud prior to nuclear division. Asymmetric mRNA localization depends upon factors that facilitate ASH1 mRNA localization (e.g., the 3' untranslated region, She proteins 1 to 5, Puf6, actin cytoskeleton, and a physical association with She2). mRNA placement precedes protein enrichment and subsequent bud emergence, implying that mRNA localization contributes to polarization. Correspondingly, mRNAs encoding proteins which are not asymmetrically distributed (i.e., Snc1, Mso1, Tub1, Pex3, and Oxa1) are not polarized. Finally, mutations which affect cortical endoplasmic reticulum (ER) entry and anchoring in the bud (myo4Delta, sec3Delta, and srp101) also affect asymmetric mRNA localization. Bud-localized mRNAs, including ASH1, were found to cofractionate with ER microsomes in a She2- and Sec3-dependent manner; thus, asymmetric mRNA transport and cortical ER inheritance are connected processes in yeast.